Application of heteroduplex analysis for CALR mutation screening detection in patients with Ph-myeloproliferative neoplasms

Background. In accordance with the World health organization clinical guidelines, the analysis of somatic mutations in the CALR gene, as well as mutations in the JAK2 and MPL genes, are included in the list of criteria for the Ph-myeloproliferative neoplasms diagnosis.More than 50 different mutation variants have been found in the CALR gene, among which the most frequent are a 52 bp deletion (c.1092_1143del), also called type 1, and a 5 bp insertion (c.1154_1155insTTGTC), also called type 2 (88 %).The remaining 12 % are other type less frequent indels or combinations thereof.It is most convenient to use sequencing methods to identify all possible variants of CALR mutations. It is also important to develop inexpensive screening test that can detect any mutations in the analyzed DNA fragment of CALR gene. This method can be heteroduplex analysis followed by electrophoresis on polyacrylamide gel (PAGE).The objective: to develop and demonstrate the feasibility of using heteroduplex analysis with separation of the PCR product by electrophoresis on non-denaturing PAGE for the CALR exon 9 mutations detection as the screening test. Materials and methods. DNA samples of 13 CALR-positive patients with different phenotypic variants of Ph-myeloproliferative neoplasms were screened by heteroduplex analysis. For the most common variants of CALR mutations (c.1092_1143del and c.1154_1155insTTGTC), a threshold determination of the mutant allele presence was analyzed.Nucleotide sequence of exon 9 fragment was determined using Sanger sequencing. Also, all 13 samples were analyzed using the pyrosequencing method to assess the allelic burden level.Results. Heteroduplex analysis revealed mutations in exon 9 of the CALR gene in all 13 patients. The threshold determinations of the method in the case of the c.1154_1155insTTGTC and c.1092_1143del analysis are 6.25 % and 3.13 % of the mutant allele presence in the patient sample, respectively.Conclusion. The proposed variant of the heteroduplex analysis with separation of the PCR product by electrophoresis on non-denaturing PAGE can be recommended for use as the preliminary screening test which is carried out before the confirming sequencing methods for the different indels (or combinations thereof) CALR mutations determine.The presence of heteroduplexes indicates the presence of a mutation, even if the mutant product is not visualized (in case of small mutations)

Using the Minor Variant Finder software to identify and quantify the allelic burden level of somatic mutations in oncohematologic diseases

Background. There are problems related to both quantitative assessment of an allele burden level of a mutant gene and interpretation of results in DNA samples with the burden level of the mutant allele less than 15–20 %, when using Sanger sequencing for analyzing somatic mutations. Applied Biosystems (USA) has developed new software Minor Variant Finder, which allows determining mutations with the allele burden level from 5 %.The objective: to determine the allele burden level and identification of minor variants of somatic mutations in the ASXL1, JAK2 genes and BCR-ABL oncogene using Minor Variant Finder software in patients with myeloproliferative neoplasms.Materials and methods. The level of mutant allele burden for 15 patients with myeloproliferative neoplasms was determined by the identified mutations using the Minor Variant Finder software, after analysis of point somatic mutations in the ASXL1, JAK2 genes and BCR-ABL oncogene by Sanger sequencing.Results. The allele burden level in all 5 ASXL1-positive samples and BCR-ABL-positive sample was determined as higher than 20 % using the Minor Variant Finder software. The allele burden level in 2 cases was higher than 20 % and in 7 cases lower than 20 %, when we analyzed 9 JAK2-positive samples.Conclusion. Minor Variant Finder software can be used to estimate the allele burden level and to identify minor variants of somatic mutations in the ASXL, JAK2 and BCR-ABL genes

ROS-generation and cellular uptake behavior of amino-silica nanoparticles arisen from their uploading by both iron-oxides and hexamolybdenum clusters

© 2020 Elsevier B.V. The present work introduces combination of superparamagnetic iron oxides (SPIONs) and hexamolybdenum cluster ([{Mo6I8}I6]2−) units within amino-decorated silica nanoparticles (SNs) as promising design of the hybrid SNs as efficient cellular contrast and therapeutic agents. The heating generated by SNs doped with SPIONs (Fe3O4@SNs) under alternating magnetic field is characterized by high specific absorption rate (SAR = 446 W/g). The cluster units deposition onto both Fe3O4@SNs and “empty” silica nanoparticles (SNs) results in Fe3O4@SNs[{Mo6I8}I6] and SNs[{Mo6I8}I6] with red cluster-centered luminescence and ability to generate reactive oxygen species (ROS) under the irradiation. The monitoring of spin-trapped ROS by ESR spectroscopy technique indicates that the ROS-generation decreases in time for SNs[{Mo6I8}I6] and [{Mo6I8}I6]2− in aqueous solutions, while it remains constant for Fe3O4@SNs[{Mo6I8}I6]. The cytotoxicity is low for both Fe3O4@SNs[{Mo6I8}I6] and SNs[{Mo6I8}I6], while the flow cytometry indicates preferable cellular uptake of the former versus the latter type of the nanoparticles. Moreover, entering into nucleus along with cytoplasm differentiates the intracellular distribution of Fe3O4@SNs[{Mo6I8}I6] from that of SNs[{Mo6I8}I6], which remain in the cell cytoplasm only. The exceptional behavior of Fe3O4@SNs[{Mo6I8}I6] is explained by residual amounts of iron ions at the silica surface